Preparation method of multilayer sandwich type epoxy magnetic microspheres and application thereof

Abstract

The application provides a preparation method of a multi-layer sandwich type epoxy group modified magnetic microsphere and application thereof. The multi-layer sandwich type epoxy magnetic microsphere prepared by the preparation method provided in the application can realize efficient and accurate cell sorting through coupling of an antibody, is suitable for cell sorting related scenes such as stem cell separation, tumor cell detection, immune cell treatment (such as CAR-T cell preparation) and pathogenic microorganism enrichment, and provides key material support for cell sorting technology in life science research and clinical diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of magnetic material preparation technology, specifically relating to a method for preparing multilayer sandwich-type epoxy group-modified magnetic microspheres and their applications. These multilayer sandwich-type epoxy magnetic microspheres can achieve efficient and precise cell sorting through antibody conjugation, making them suitable for cell sorting-related scenarios such as stem cell isolation, tumor cell detection, immunotherapy (e.g., CAR-T cell preparation), and pathogen enrichment. They provide key material support for cell sorting technologies in life science research and clinical diagnosis. Background Technology

[0002] Cell sorting is a key technology in life science research and clinical diagnosis and treatment, widely used in stem cell isolation, tumor cell detection, immunotherapy, and pathogen enrichment. Efficient and precise cell sorting technologies not only advance the exploration of basic biological mechanisms but also provide crucial support for precision medicine (such as circulating tumor cell (CTC) detection and CAR-T cell preparation). Currently, mainstream cell sorting technologies include flow cytometry (FACS), density gradient centrifugation, microfluidic chips, and magnetic microsphere sorting. Among these, magnetic microsphere sorting technology has become a research hotspot and mainstream application in the biomedical field due to its advantages such as ease of operation, non-invasiveness, scalability, and applicability to complex samples (such as whole blood and tissue homogenates).

[0003] Among various types of modified magnetic microspheres, epoxy-modified magnetic microspheres exhibit a mild reaction during antibody labeling, requiring no activation reagents and minimizing cell damage, thus expanding their application range. However, existing epoxy-modified magnetic microspheres generally suffer from poor dispersibility due to their simple structure, uneven magnetic core loading affecting magnetic response speed, or low antibody binding efficiency due to insufficient surface epoxy group density, making it difficult to fully meet the practical needs of high-precision and high-efficiency cell sorting. Therefore, developing an epoxy-modified magnetic microsphere with a superior structure and more stable performance has become crucial to overcoming current technological bottlenecks. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing multilayer sandwich-type epoxy magnetic microspheres (magnetic beads), the method comprising the following steps: (1) Preparation of polyglycidyl methacrylate (PGMA) seed microspheres: polyglycidyl methacrylate (PGMA) seed microspheres were prepared from the monomer glycidyl methacrylate (GMA); (2) Preparation of PGMA-NH2 microspheres: The PGMA seed microspheres prepared in step (1) are combined with polyamines to prepare PGMA-NH2 microspheres; (3) Preparation of magnetic PGMA-NH2 microspheres: The PGMA-NH2 microspheres prepared in step (2) are further prepared into magnetic PGMA-NH2 microspheres; (4) Preparation of magnetic alkenyl microspheres: The magnetic PGMA-NH2 microspheres prepared in step (3) are reacted with glycidyl methacrylate (GMA) to obtain magnetic alkenyl microspheres; (5) Preparation of multilayer sandwich carboxyl magnetic microspheres: The magnetic alkenyl microspheres prepared in step (4) are reacted with acrylic acid to obtain the multilayer sandwich carboxyl magnetic microspheres; (6) Preparation of multilayer sandwich amino magnetic microspheres: The multilayer sandwich carboxyl magnetic microspheres prepared in step (5) are reacted with polyamines to obtain the multilayer sandwich amino magnetic microspheres; (7) Preparation of multilayer sandwich alkenyl magnetic microspheres: The multilayer sandwich amino magnetic microspheres prepared in step (6) are reacted with methacrylic anhydride to obtain multilayer sandwich alkenyl magnetic microspheres; (8) Preparation of multilayer sandwich epoxy magnetic microspheres: The multilayer sandwich alkenyl magnetic microspheres prepared in step (7) are reacted with glycidyl methacrylate (GMA) to obtain the multilayer sandwich epoxy magnetic microspheres.

[0005] According to an embodiment of the present invention, in step (1), the polyglycidyl methacrylate (PGMA) seed microspheres are a polyglycidyl methacrylate (PGMA) seed microsphere suspension.

[0006] According to an embodiment of the present invention, step (1) specifically involves: preparing PGMA seed microspheres by free radical polymerization of the monomer glycidyl methacrylate (GMA) in the presence of an initiator and a dispersant.

[0007] According to an embodiment of the present invention, in step (1), the initiator is selected from at least one of inorganic peroxide initiators and organic azo initiators; preferably, the inorganic peroxide initiator is selected from at least one of potassium persulfate, ammonium persulfate, sodium persulfate, etc.; preferably, the organic azo initiator is selected from at least one of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, etc. Exemplarily, in step (1), the initiator is selected from azobisisobutyronitrile (AIBN).

[0008] According to an embodiment of the present invention, in step (1), the dispersant is selected from one, two or more of the following: sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, polyvinylpyrrolidone or polyvinyl alcohol, etc. Exemplarily, in step (1), the dispersant is selected from polyvinylpyrrolidone.

[0009] According to an embodiment of the present invention, in step (1), the volume-to-mass ratio of the glycidyl methacrylate and the dispersant is 10-40 mL: 5-20 g, preferably 20-40 mL: 5-15 g, for example 30 mL: 8 g.

[0010] According to an embodiment of the present invention, in step (1), the volume-to-mass ratio of the glycidyl methacrylate to the initiator is 10-40 mL:0.1-1 g, preferably 20-40 mL:0.2-0.8 g, for example 30 mL:0.5 g.

[0011] According to an embodiment of the present invention, step (1) is carried out in a solvent; preferably, the solvent is selected from at least one of water and ethanol. Preferably, the mass concentration (g / mL) of glycidyl methacrylate (GMA) in the solvent is 5-20%, more preferably 10-20%, for example 10%.

[0012] According to an embodiment of the present invention, step (1) is carried out under an inert atmosphere.

[0013] According to an embodiment of the present invention, in step (1), the reaction temperature is 60-85°C, for example 70°C; and / or the reaction time is 4-48h, for example 24h.

[0014] According to an embodiment of the present invention, step (1) specifically comprises: dissolving the dispersant in a solvent, then adding glycidyl methacrylate, and heating and stirring under a nitrogen atmosphere; then adding an initiator to the resulting mixture, and heating and stirring under a nitrogen atmosphere; after the reaction is completed, a monodisperse PGMA seed microsphere suspension is obtained. According to an embodiment of the present invention, the heating and stirring time is 10 min-60 min, for example 30 min; and / or, the heating and stirring temperature is 60-85°C, for example 70°C. According to an embodiment of the present invention, the heating and stirring reaction temperature is 60-85°C, for example 70°C; and / or, the heating and stirring reaction time is 4-48 h, for example 24 h.

[0015] According to an embodiment of the present invention, in step (2), the polyamine is selected from one, two, or more of the following: ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polyethyleneimine, etc. For example, the polyamine in step (2) is ethylenediamine.

[0016] According to an embodiment of the present invention, in step (2), the mass-to-volume ratio of PGMA seed microspheres to polyamine is 1 g:(10-200) mL, 1 g:(10-150) mL, for example, 1 g:100 mL. According to an embodiment of the present invention, step (2) specifically involves: mixing and reacting the PGMA seed microspheres prepared in step (1) with polyamine, water, and surfactant to prepare PGMA-NH2 microspheres; preferably, further mixing and reacting the prepared PGMA-NH2 microspheres with polyamine and crosslinking agent (forming a dense amino surface, amino modification reaction) to obtain the final PGMA-NH2 microspheres. According to an embodiment of the present invention, step (2) specifically involves: mixing and reacting the PGMA seed microspheres prepared in step (1) with polyamine, water, and surfactant (one-step swelling and amination) to prepare PGMA-NH2 microspheres; then further mixing and reacting the prepared PGMA-NH2 microspheres with polyamine, water, and crosslinking agent (increasing surface density, crosslinking reaction) to obtain the final PGMA-NH2 microspheres.

[0017] According to an embodiment of the present invention, in step (2), the particle size of the PGMA-NH2 microspheres and the final PGMA-NH2 microspheres is 2-4 μm.

[0018] According to an embodiment of the present invention, in step (2) of the preparation of PGMA-NH2 microspheres and in the final preparation of PGMA-NH2 microspheres, the polyamines are the same or different, and are independently selected from one, two or more of the following: ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polyethyleneimine, etc. For example, the polyamine in step (2) is ethylenediamine.

[0019] According to an embodiment of the present invention, in step (2), the crosslinking agent is selected from one, two, or more of the following: epichlorohydrin, 1,4-butanediol diglycidyl ether, glycerol triglycidyl ether, ethylene glycol diglycidyl ether, etc. For example, the crosslinking agent in step (2) is epichlorohydrin.

[0020] According to an embodiment of the present invention, in step (2), the surfactant is selected from one, two, or more of the following: sodium 4-methyl-N-[(dodecylamino)carbonyl]benzenesulfonamide, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium dodecyl sulfonate, etc. Exemplarily, the surfactant in step (2) is sodium dodecyl sulfate.

[0021] According to an embodiment of the present invention, in step (2) during the preparation of PGMA-NH2 microspheres, the mass-volume ratio of PGMA seed microspheres to polyamine is 1 g:(10-200) mL, 1 g:(10-150) mL, for example 1 g:100 mL.

[0022] According to an embodiment of the present invention, in step (2), the mass ratio of PGMA seed microspheres to surfactant is 1:(0.1-2), 1:(0.1-1.5), for example 1:1.

[0023] According to an embodiment of the present invention, in step (2) the final preparation of PGMA-NH2 microspheres, the mass-volume ratio of PGMA seed microspheres to polyamine is 1 g:(1-20) mL, 1 g:(1-15) mL, for example 1 g:10 mL.

[0024] According to an embodiment of the present invention, in step (2) the final preparation of PGMA-NH2 microspheres, the mass-volume ratio of PGMA seed microspheres and crosslinking agent is 1 g: (0.1-10) mL, 1 g: (0.1-1) mL, for example 1 g: 10 mL.

[0025] According to an embodiment of the present invention, in step (2) during the preparation of PGMA-NH2 microspheres, the reaction temperature is 75-85℃, for example 80℃; and / or the reaction time is 10-24h, for example 20h.

[0026] According to an embodiment of the present invention, in step (2) the final preparation of PGMA-NH2 microspheres, the reaction temperature is 15-60℃, for example 25℃; and / or the reaction time is 2-24h, for example 12h.

[0027] According to an embodiment of the present invention, step (2) further includes a post-processing step; preferably, the post-processing step includes centrifugation, washing, drying and other steps; preferably, the post-processing step specifically involves: centrifugation after the reaction is completed, washing of the solid product, vacuum drying, and obtaining PGMA-NH2 microspheres.

[0028] According to an embodiment of the present invention, step (3) specifically involves adding the PGMA-NH2 microspheres prepared in step (2) to a Fe-containing... 2+ Compounds and Fe 3+ Ammonia was added to an aqueous solution of the compound to react and obtain magnetic PGMA-NH2 microspheres.

[0029] According to an embodiment of the present invention, in step (3), the reaction temperature is 75-85°C, for example 75°C; and / or the reaction time is 0.5-2h, for example 0.5h.

[0030] According to an embodiment of the present invention, in step (3), the Fe-containing 2+ The compound is selected from ferrous salts (known in the art), such as at least one selected from ferrous sulfate, ferrous sulfate heptahydrate, ferrous ammonium sulfate, ferrous chloride, ferrous chloride tetrahydrate (FeCl2·4H2O), etc.

[0031] According to an embodiment of the present invention, in step (3), the Fe-containing 3+ The compound is selected from iron salts (known in the art), such as at least one selected from ferric sulfate, ferric nitrate, ferric chloride, ferric chloride hexahydrate (FeCl3·6H2O), etc.

[0032] According to an embodiment of the present invention, in step (3), the Fe-containing 2+ Compounds and Fe-containing compounds 3+ The molar ratio of the compounds is 1:1 to 2; for example, it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.

[0033] According to an embodiment of the present invention, in step (3), PGMA-NH2 microspheres and Fe-containing... 3+ The mass ratio of the compounds is 1:0.1-0.5, for example 1:0.1, 1:0.2, 1:0.25, 1:0.3, 1:0.4, 1:0.5.

[0034] According to an embodiment of the present invention, in step (3), the amount of ammonia added is not particularly limited, as long as the pH of the reaction system is 10-11. According to an embodiment of the present invention, in step (3), after adding ammonia, the pH of the reaction system is 10-11. According to an embodiment of the present invention, in step (3), the mass-to-volume ratio of PGMA-NH2 microspheres to ammonia is 1 g : 10-30 mL, for example, 1 g : 20 mL.

[0035] According to an embodiment of the present invention, step (3) further includes a post-processing step; preferably, the post-processing step includes steps such as recovery (e.g., magnetic recovery, centrifugal recovery), washing, and drying; preferably, the post-processing step specifically involves: cooling after the reaction is completed, recovery, ultrasonic washing, vacuum drying, and obtaining magnetic PGMA-NH2 microspheres.

[0036] According to an embodiment of the present invention, step (4) specifically involves: mixing and reacting the magnetic PGMA-NH2 microspheres prepared in step (3) with an organic solvent and glycidyl methacrylate to obtain magnetic alkenyl microspheres.

[0037] According to an embodiment of the present invention, in step (4), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran, ethylene glycol, diethylene glycol, diethylene glycol dimethyl ether, etc. In step (4) of the present invention, the amount of organic solvent added is not particularly limited, as long as it is sufficient to dissolve the raw materials. According to an embodiment of the present invention, in step (4), the mass-to-volume ratio of the magnetic PGMA-NH2 microspheres to the organic solvent is, for example, 1 g: 20-80 mL, for example, 1 g: 50 mL.

[0038] According to an embodiment of the present invention, in step (4), the mass-to-volume ratio of magnetic PGMA-NH2 microspheres to GMA is 0.01-0.5 g:1 mL, preferably 0.01-0.3 g:1 mL; exemplary, it is 0.1 g:1 mL, 1 g:10 mL, 0.01 g / mL, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL or 0.1 g / mL.

[0039] According to an embodiment of the present invention, in step (4), the reaction temperature is 75-85°C, for example 75°C; and / or the reaction time is 10-24 h, for example 16 h.

[0040] According to an embodiment of the present invention, step (4) further includes a post-processing step; preferably, the post-processing step includes steps such as recovery (e.g., magnetic recovery, centrifugal recovery), washing, and drying; preferably, the post-processing step specifically involves: after the reaction is completed, the product is recovered, the solid product is washed, and vacuum dried to obtain magnetic alkenyl microspheres.

[0041] According to an embodiment of the present invention, step (5) specifically involves: mixing and reacting the magnetic alkenyl microspheres prepared in step (4) with acrylic acid, organic solvent and initiator to obtain the multilayer sandwich-type carboxyl magnetic microspheres.

[0042] According to an embodiment of the present invention, in step (5), the mass-to-volume ratio of magnetic alkenyl microspheres to acrylic acid is 1 g: 1-20 mL, for example, 1 g: 10 mL.

[0043] According to an embodiment of the present invention, in step (5), the volume-to-mass ratio of acrylic acid and initiator is 1-50 mL: 1 g, for example 20 mL: 1 g or 10 mL: 0.5 g.

[0044] According to an embodiment of the present invention, in step (5), the initiator is selected from at least one of inorganic peroxide initiators and organic azo initiators; preferably, the inorganic peroxide initiator is selected from at least one of potassium persulfate, ammonium persulfate, sodium persulfate, etc.; preferably, the organic azo initiator is selected from at least one of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, etc. Exemplarily, in step (5), the initiator is selected from azobisisobutyronitrile (AIBN).

[0045] According to an embodiment of the present invention, in step (5), the reaction temperature is 70-85°C, for example 75°C; and / or the reaction time is 10-24 h, for example 16 h.

[0046] According to an embodiment of the present invention, in step (5), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran, ethylene glycol, diethylene glycol, diethylene glycol dimethyl ether, etc. In step (5) of the present invention, the amount of organic solvent added is not particularly limited, as long as the raw materials are uniformly dispersed or dissolved. According to an embodiment of the present invention, in step (5), the mass-to-volume ratio of the magnetic alkenyl microspheres to the organic solvent is, for example, 1 g: 20-200 mL, for example, 1 g: 100 mL.

[0047] According to an embodiment of the present invention, step (5) further includes a post-processing step; preferably, the post-processing step includes steps such as recovery (e.g., magnetic recovery, centrifugal recovery), washing, and drying; preferably, the post-processing step specifically involves: after the reaction is completed, recovery, washing, and vacuum drying to obtain the multilayer sandwich-type carboxyl magnetic microspheres.

[0048] According to an embodiment of the present invention, step (6) specifically involves: the magnetic carboxyl microspheres prepared in step (5) are mixed and reacted with a polyamine, a crosslinking agent (e.g., EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)), and an amino protecting agent (e.g., NHS (N-hydroxysuccinimide)) in a buffer solution to obtain the multilayer sandwich-type amino magnetic microspheres. According to an embodiment of the present invention, step (6) specifically involves: the magnetic carboxyl microspheres prepared in step (5), the crosslinking agent, and the amino protecting agent are dispersed in a buffer solution, stirred, and separated to obtain magnetic beads; then the magnetic beads are dispersed in a buffer solution, and a polyamine is added to react, resulting in the multilayer sandwich-type amino magnetic microspheres.

[0049] According to an embodiment of the present invention, in step (6), the buffer solution is a MES buffer solution; preferably, the pH is 4.0-7.0; preferably, the concentration of the MES buffer solution is 0.1M-0.5M, for example, 0.25M. According to an embodiment of the present invention, in step (6), the mass-to-volume ratio of the magnetic carboxyl microspheres or magnetic beads to the buffer solution is 1 g: 50-300 mL, for example, 1 g: 200 mL.

[0050] According to an embodiment of the present invention, in step (6), the mass-to-volume ratio of the multilayer sandwich-type carboxylated magnetic microspheres to the polyamine is 1 g: 1-100 mL, preferably 1 g: 1-50 mL, 1 g: 20-80 mL, for example 1 g: 50 mL.

[0051] According to an embodiment of the present invention, in step (6), the polyamine is one or more mixtures selected from ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polyethyleneimine, etc. For example, the polyamine in step (6) is tetraethylenepentamine.

[0052] According to an embodiment of the present invention, in step (6), the reaction temperature is room temperature; and / or the reaction time is 10-24h, for example 10h or 12h.

[0053] According to an embodiment of the present invention, step (6) further includes a post-processing step; preferably, the post-processing step includes steps such as recovery (e.g., magnetic recovery, centrifugal recovery), washing, and drying; preferably, the post-processing step specifically involves: after the reaction is completed, recovering the magnetic beads, washing, and vacuum drying to obtain the multilayer sandwich-type amino magnetic microspheres.

[0054] According to an embodiment of the present invention, step (7) specifically involves: mixing and reacting the multilayer sandwich-type amino magnetic microspheres prepared in step (6) with an organic solvent and acrylic anhydride to obtain multilayer sandwich-type alkenyl magnetic microspheres.

[0055] According to an embodiment of the present invention, the acrylic anhydride is methacrylic anhydride.

[0056] According to an embodiment of the present invention, in step (7), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran, ethylene glycol, diethylene glycol, diethylene glycol dimethyl ether, etc. In step (7) of the present invention, the amount of organic solvent added is not particularly limited, as long as it is sufficient to dissolve or uniformly disperse the raw materials. According to an embodiment of the present invention, in step (7), the mass-to-volume ratio of the multilayer sandwich-type amino magnetic microspheres to the organic solvent is 1 g: 20-200 mL, for example, 1 g: 100 mL.

[0057] According to an embodiment of the present invention, in step (7), the mass-to-volume ratio of the multilayer sandwich-type amino magnetic microspheres and acrylic anhydride is 1g:1-50 mL, for example, 1g:10 mL.

[0058] According to an embodiment of the present invention, in step (7), the reaction temperature is 70-85°C, for example 75°C; and / or the reaction time is 10-24 h, for example 20 h.

[0059] According to an embodiment of the present invention, step (7) further includes a post-processing step; preferably, the post-processing step includes steps such as recovery (e.g., magnetic recovery, centrifugal recovery), washing, and drying; preferably, the post-processing step specifically involves: after the reaction is completed, recovering the magnetic beads, washing, and vacuum drying to obtain the multilayer sandwich alkenyl magnetic microspheres.

[0060] According to an embodiment of the present invention, step (8) specifically involves: mixing and reacting the multilayer sandwich-type alkenyl magnetic microspheres prepared in step (7) with glycidyl methacrylate, an organic solvent and an initiator to obtain the multilayer sandwich-type epoxy magnetic microspheres.

[0061] According to an embodiment of the present invention, in step (8), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran, ethylene glycol, diethylene glycol, diethylene glycol dimethyl ether, etc. In step (8) of the present invention, the amount of organic solvent added is not particularly limited, as long as it is sufficient to dissolve or uniformly disperse the raw materials. According to an embodiment of the present invention, in step (8), the mass-to-volume ratio of the multilayer sandwich-type alkenyl magnetic microspheres to the organic solvent is 1 g: 20-200 mL, for example, 1 g: 100 mL.

[0062] According to an embodiment of the present invention, in step (8), the initiator is selected from at least one of inorganic peroxide initiators and organic azo initiators; preferably, the inorganic peroxide initiator is selected from at least one of potassium persulfate, ammonium persulfate, sodium persulfate, etc.; preferably, the organic azo initiator is selected from at least one of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, etc. Exemplarily, in step (8), the initiator is selected from azobisisobutyronitrile (AIBN).

[0063] According to an embodiment of the present invention, in step (8), the mass-to-volume ratio of the multilayer sandwich alkenyl magnetic microspheres and glycidyl methacrylate (GMA) is 1g:1-50 mL, preferably 1g:2-30 mL, for example 1g:10 mL.

[0064] According to an embodiment of the present invention, in step (8), the mass ratio of the multilayer sandwich alkenyl magnetic microspheres to the initiator is 1:0.01-1, preferably 1:0.1-0.8, for example 1:0.5.

[0065] According to an embodiment of the present invention, in step (8), the reaction temperature is 70-85°C, for example 75°C; and / or the reaction time is 10-24 h, for example 20 h.

[0066] According to an embodiment of the present invention, step (8) further includes a post-processing step; preferably, the post-processing step includes steps such as recovery (e.g., magnetic recovery, centrifugal recovery), washing, and drying; preferably, the post-processing step specifically involves: after the reaction is completed, recovering the magnetic beads, washing, and vacuum drying to obtain the multilayer sandwich epoxy magnetic microspheres.

[0067] According to an embodiment of the present invention, the preparation method includes the following steps: (1) Preparation of polyglycidyl methacrylate (PGMA) seed microsphere suspension: PGMA seed microsphere suspension was prepared by free radical polymerization of monomer glycidyl methacrylate (GMA) in the presence of initiator and crosslinking agent; (2) Preparation of PGMA-NH2 microspheres: The PGMA seed microsphere suspension prepared in step (1) is mixed with polyamine, water and surfactant and reacted to swell and amination in one step to prepare amino microspheres; then the obtained amino microspheres are mixed with polyamine, water (e.g. deionized water) and crosslinking agent to obtain PGMA-NH2; (3) Preparation of magnetic PGMA-NH2 microspheres: The PGMA-NH2 microspheres prepared in step (2) are added to Fe-containing... 2+ Compounds and Fe 3+ Ammonia was added to an aqueous solution of the compound to react and obtain magnetic PGMA-NH2 microspheres. (4) Preparation of magnetic alkenyl microspheres: The magnetic PGMA-NH2 microspheres prepared in step (3) are mixed with organic solvent and glycidyl methacrylate to obtain magnetic alkenyl microspheres; (5) Preparation of multilayer sandwich carboxyl magnetic microspheres: The magnetic alkenyl microspheres prepared in step (4) are mixed and reacted with acrylic acid, organic solvent and initiator to obtain the multilayer sandwich carboxyl magnetic microspheres; (6) Preparation of multilayer sandwich amino magnetic microspheres: The magnetic carboxyl microspheres prepared in step (5) are mixed and reacted with polyamine, EDC and NHS in a buffer solution (e.g., MES system) to obtain the multilayer sandwich amino magnetic microspheres; (7) Preparation of multilayer sandwich alkenyl magnetic microspheres: The multilayer sandwich amino magnetic microspheres prepared in step (6) are mixed with organic solvent and acrylic anhydride to react and obtain multilayer sandwich alkenyl magnetic microspheres; (8) Preparation of multilayer sandwich epoxy magnetic microspheres: The multilayer sandwich alkenyl magnetic microspheres prepared in step (7) are mixed and reacted with glycidyl methacrylate, organic solvent and initiator to obtain the multilayer sandwich epoxy magnetic microspheres.

[0068] According to an embodiment of the present invention, the preparation method of the multilayer sandwich epoxy magnetic microspheres specifically includes the following steps: (1) Preparation of PGMA seed microsphere suspension: Polyvinylpyrrolidone was dissolved in anhydrous ethanol, GMA was added, and the mixture was stirred for 10-30 min under a nitrogen atmosphere and heated to 65-85℃. An initiator (e.g., AIBN) was added to the resulting mixture, and the mixture was stirred at 200-400 rpm and reacted at 60℃-85℃ for 4-24 h under a nitrogen atmosphere. After the reaction was completed, a PGMA seed microsphere suspension was obtained. (2) Preparation of PGMA-NH2 microspheres PGMA seed microsphere suspension was dispersed in water containing polyamine, surfactant was added, and the reaction was carried out at 75-85℃ for 10-24h. After the reaction was completed, the product was centrifuged, washed, and vacuum dried to obtain amino microspheres. The obtained amino microspheres were mixed with polyamine, water, and crosslinking agent, stirred at 25℃ for 8-20h, and centrifuged after the reaction was completed. The product was washed, vacuum dried, and PGMA-NH2 microspheres were obtained. (3) Preparation of magnetic PGMA-NH2 microspheres: Take the PGMA-NH2 microspheres prepared in step (2) and add them to a Fe-containing... 2+ Compounds and Fe 3+ In an aqueous solution of a compound (e.g., FeCl3 and FeCl2, such as FeCl3·6H2O and FeCl2·4H2O), the mixture was stirred at room temperature for 15-45 min, then heated to 75-85 °C, ammonia was added, and the reaction was allowed to proceed for 0.5-2 h. After the reaction was completed, the mixture was cooled, centrifuged, and the solid product was washed and vacuum dried to obtain magnetic PGMA-NH2 microspheres. (4) Preparation of magnetic alkenyl microspheres Take the magnetic PGMA-NH2 microspheres prepared in step (3) and mix them with an organic solvent. Heat the mixture to 70-85℃, then add GMA and react for 10-24 h. After the reaction is complete, centrifuge the mixture, wash the solid product and dry it under vacuum to obtain magnetic alkenyl microspheres. (5) Preparation of multilayer sandwich-type carboxyl magnetic microspheres Take the magnetic alkenyl microspheres prepared in step (4), and disperse them with acrylic acid and an initiator (e.g., AIBN) in an organic solvent. Stir and react at 75-85℃ for 10-24h. After the reaction is completed, magnetic products are separated by magnetic adsorption to obtain multilayer sandwich carboxyl magnetic microspheres. (6) Preparation of multilayer sandwich-type amino magnetic microspheres The multilayer sandwich-type carboxyl magnetic microspheres prepared in step (5) are dispersed in a buffer solution, and a crosslinking agent (e.g., EDC) and an amino protectant (e.g., NHS) are added. The mixture is stirred at room temperature for 2-12 h, and then a polyamine is added and stirred at room temperature for 2-16 h. After the reaction is completed, the magnetic product is separated and washed to obtain multilayer sandwich-type amino magnetic microspheres. (7) Preparation of multilayer sandwich alkenyl magnetic microspheres Take the multilayer sandwich amino magnetic microspheres prepared in step (6) and acrylic anhydride, disperse them in an organic solvent, stir at 50-85℃ for 8-24 h, separate the magnetic product after the reaction, and obtain multilayer sandwich alkenyl magnetic microspheres after washing. (8) Preparation of multilayer sandwich epoxy magnetic microspheres Take the multilayer sandwich alkenyl magnetic microspheres prepared in step (7), GMA, and initiator (e.g., AIBN) and disperse them in an organic solvent. Stir at 50-85℃ for 8-24 h. After the reaction is complete, magnetic products are separated by magnetic adsorption and washed to obtain multilayer sandwich epoxy magnetic microspheres.

[0069] According to an embodiment of the present invention, the Z-average particle size of the multilayer sandwich epoxy magnetic microspheres is 1000 nm-8000 nm, for example, 4000 nm. According to an embodiment of the present invention, the saturation magnetic strength (VSM) of the multilayer sandwich epoxy magnetic microspheres is 30 emu / g-50 emu / g, for example, 42 emu / g. According to an embodiment of the present invention, the magnetic attraction time (e.g., in aqueous solution) of the multilayer sandwich epoxy magnetic microspheres is 1 s-5 s, for example, 2 s.

[0070] According to an embodiment of the present invention, the effective functional group content on the surface of the multilayer sandwich epoxy magnetic microspheres is 1.0^10. -11 μmol / beads -6.0^10 -11 μmol / beads, for example 2.0^10 -11 μmol / beads, 2.5^10 -11 μmol / beads, 3.0^10 -11 μmol / beads, 3.5^10 -11 μmol / beads, 4.0^10 -11 μmol / beads, 4.5^10 -11 μmol / beads, 5.0^10-11 μmol / beads.

[0071] In this invention, the preparation route or reaction mechanism of multilayer sandwich epoxy magnetic microspheres is as follows: Figure 1 As shown.

[0072] The present invention also provides multilayer sandwich epoxy magnetic microspheres (magnetic beads) prepared according to the above preparation method.

[0073] The present invention also provides antibody-labeled multilayer sandwich epoxy magnetic microspheres (magnetic beads).

[0074] According to an embodiment of the present invention, the antibody is, for example, a CD3 / CD28 antibody.

[0075] The present invention also provides the application of the multilayer sandwich epoxy magnetic microspheres prepared according to the above preparation method, the multilayer sandwich epoxy magnetic microspheres, and the antibody-labeled multilayer sandwich epoxy magnetic microspheres in cell sorting, protein / antibody separation and purification, nucleic acid separation and purification, and immunoassay / detection, or in the preparation of reagents for cell sorting, protein / antibody separation and purification, nucleic acid separation and purification, and immunoassay / detection.

[0076] According to the embodiments of the present invention, the application scenarios of cell sorting include stem cell isolation, tumor cell detection, immune cell therapy (such as CAR-T cell preparation), and pathogenic microorganism enrichment.

[0077] The beneficial effects of this invention are: Traditional epoxy magnetic microspheres typically use polystyrene (PS) microspheres as a substrate. Larger PS particles are obtained through porous swelling polymerization, followed by nitration-reduction modification of the magnetic bead surface with amino groups. Magnetization then yields magnetic amino microspheres (PS-NH2). These are further modified with monomers containing two or more epoxy groups to obtain epoxy-modified magnetic microspheres. The polymerization mechanism is as follows:

[0078]

[0079] Magnetic beads prepared in this way will have unreduced nitro groups on their surface. Nitro groups have certain cytotoxicity, which can reduce cell viability, induce apoptosis, or inhibit cell proliferation.

[0080] Patent application CN117024788A first prepares PGMA microspheres, then modifies the surface with amino groups, magnetizes them by co-precipitation, and finally reacts the surface amino groups with monomers containing two epoxy groups to obtain epoxy group-modified magnetic microspheres.

[0081]

[0082] In this step, the co-precipitated iron oxide is attached to the surface of the microspheres. It is difficult to completely coat the iron oxide in the subsequent epoxy modification step, and the exposed iron oxide will affect the application of the magnetic beads.

[0083] Patent application CN114736402A discloses a method for preparing epoxy magnetic beads. The method first prepares a magnetic core, then mixes the magnetic core with a polymer monomer containing epoxy groups, and directly encapsulates the mixture through dispersion polymerization to form epoxy group-modified magnetic microspheres.

[0084]

[0085] Although this method involves fewer steps, the magnetic beads produced are small in size, making it impossible to obtain micron-sized magnetic beads, and the size of the magnetic beads is not uniform.

[0086] This invention provides a method for preparing multilayer sandwich-type epoxy group-modified magnetic microspheres and their applications. The multilayer sandwich-type epoxy magnetic microspheres prepared according to the method of this invention can achieve efficient and precise cell sorting through antibody conjugation. They are suitable for cell sorting-related scenarios such as stem cell isolation, tumor cell detection, immunotherapy (e.g., CAR-T cell preparation), and pathogen enrichment, providing key material support for cell sorting technology in life science research and clinical diagnosis. Attached Figure Description

[0087] Figure 1 This describes the preparation route or reaction mechanism of the multilayer sandwich epoxy magnetic microspheres of the present invention.

[0088] Figure 2 This is a SEM image of the multilayer sandwich epoxy magnetic microspheres in Example 1.

[0089] Figure 3 The image shows the hysteresis loop (VSM) diagram of the multilayer sandwich epoxy magnetic microspheres in Example 1.

[0090] Figure 4 This is a flow cytometry diagram showing the activation effect of magnetic beads.

[0091] Figure 5 This is a flow cytometry plot showing the transfection efficiency after cell incubation. Detailed Implementation

[0092] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0093] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0094] Example 1: A method for preparing and characterizing multilayer sandwich epoxy magnetic microspheres. S1. Preparation of PGMA seed microsphere suspension 8 g of polyvinylpyrrolidone was dissolved in 300 mL of anhydrous ethanol, and 30 mL of glycidyl methacrylate (GMA) was added. The mixture was stirred for 30 min under a nitrogen atmosphere and heated to 70 °C. 500 mg of AIBN (azobisisobutyronitrile) was added to the resulting mixture. The mixture was stirred at 300 rpm and reacted at 70 °C for 24 h under a nitrogen atmosphere. After the reaction was completed, a PGMA seed microsphere suspension was obtained.

[0095] S2. Preparation of PGMA-NH2 microspheres 1.0 g of PGMA seed microsphere suspension was added to a mixed solution of 100 mL ethylenediamine and 50 mL deionized water, and 1 g of sodium dodecyl sulfate was added. The mixture was reacted at 80 °C for 20 h. After the reaction was completed, the mixture was centrifuged, the solid product was washed and vacuum dried, and the obtained amino microspheres were dispersed in 100 mL of deionized water. 10 mL of ethylenediamine and 10 mL of epichlorohydrin were added, and the mixture was stirred at 25 °C for 12 h to obtain PGMA-NH2 microspheres.

[0096] S3. Preparation of magnetic PGMA-NH2 microspheres 1.0 g of PGMA-NH2 microspheres prepared in step S2 were added to an aqueous solution containing 1.1 g of FeCl3·6H2O and 0.4 g of FeCl2·4H2O. The solution was heated to 75 °C, and 20 mL of ammonia water was added. The reaction was carried out for 0.5 h. After the reaction was completed, the solution was cooled, the magnetic beads were recovered by centrifugation, ultrasonically washed and vacuum dried to obtain magnetic PGMA-NH2 microspheres.

[0097] S4. Preparation of magnetic alkenyl microspheres 1.0 g of magnetic PGMA-NH2 microspheres prepared in step S3 were dispersed in 50 mL of ethanol, heated to 75 °C, and then 10 mL of GMA (glycidyl methacrylate) was added. The reaction was carried out for 16 h. After the reaction was completed, the product was centrifuged, washed, and vacuum dried to obtain magnetic alkenyl microspheres.

[0098] S5. Preparation of multilayer sandwich-type carboxyl magnetic microspheres 1.0 g of magnetic alkenyl microspheres prepared in step S4 were dispersed in 10 mL of acrylic acid and 500 mg of AIBN (azobisisobutyronitrile) in 100 mL of ethanol. The mixture was stirred at 75 °C for 16 h. After the reaction was completed, the magnetic product was separated by magnetic adsorption to obtain multilayer sandwich-type carboxyl magnetic microspheres.

[0099] S6. Preparation of multilayer sandwich-type amino magnetic microspheres Take 1 g of the multilayer sandwich-type carboxylated magnetic beads prepared in step S5, 2 g of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 2.5 g of NHS (N-hydroxysuccinimide) and disperse them in 200 mL of 0.25 M MES (2-(N-morpholine)ethanesulfonic acid) solution. Stir at room temperature for 2 h. After stirring, separate the magnetic beads by magnetic attraction.

[0100] The magnetic beads were dispersed in 200 mL of 0.25 M MES solution, and 50 mL of tetraethylenepentamine was added. The mixture was stirred at room temperature for 10 h. After stirring, the magnetic beads were separated by magnetic attraction. The solid product was washed and dried under vacuum to obtain multilayer sandwich-type amino magnetic microspheres.

[0101] S7. Preparation of multilayer sandwich-type alkenyl magnetic microspheres 1 g of the multilayer sandwich-type amino magnetic microspheres prepared in step S6 were dispersed in 100 mL of ethanol, heated to 75 °C, and then 10 mL of methacrylic anhydride was added and stirred for 20 h. After the reaction was completed, the magnetic beads were separated by magnetic attraction, and the solid product was washed and vacuum dried to obtain multilayer sandwich-type alkenyl magnetic microspheres.

[0102] S8. Preparation of multilayer sandwich epoxy magnetic microspheres 1 g of the multilayer sandwich-type alkenyl magnetic microspheres prepared in step S7 were dispersed in 100 mL of ethanol. After heating to 75 °C, 10 mL of GMA (glycidyl methacrylate) and 500 mg of AIBN (azobisisobutyronitrile) were added and stirred for 20 h. After the reaction was completed, the magnetic beads were separated by magnetic attraction. The solid product was washed and vacuum dried to obtain multilayer sandwich-type epoxy magnetic microspheres.

[0103] The physical properties of the multilayer sandwich epoxy magnetic microspheres are shown in Table 1 below.

[0104] Table 1 Physical properties of multilayer sandwich epoxy magnetic microspheres

[0105] Figure 2 The image shows the VSM diagram of a multilayer sandwich epoxy magnetic microsphere. Figure 2 It can be seen that the microspheres are superparamagnetic, with a saturation magnetic intensity of 42 emu / g.

[0106] Example 2: Detection of effective functional group content on the surface of magnetic beads Based on the magnetic bead concentration, take 4.0^10. 8 The corresponding volume of magnetic bead dispersion is placed in a 1.5 or 2 mL centrifuge tube. Magnetic adsorption is performed for the required time until the supernatant is clear and colorless. The supernatant is then removed. Add 1 mL of PBS buffer, vortex to mix, and magnetically remove the supernatant until it is clear and colorless. Repeat twice. Add 1 mL of 10 mg / mL BSA solution to the washed magnetic beads that have no supernatant, vortex to mix, and centrifuge briefly to ensure that there are no magnetic beads left on the cap. Use a rotary mixer to seal the centrifuge tube cap and incubate overnight at room temperature. After incubation, transfer the centrifuge tube to a magnetic rack and magnetically attract the supernatant until it is clear and colorless. Transfer the supernatant to a new centrifuge tube and dilute it 5 times with PBS before testing. Following the instructions of a commercially available BCA test kit, prepare the BSA standard curve solution at the following concentrations:

[0107] Referring to the instructions of the commercially available BCA test kit, the absorbance values ​​of the standard curve and the supernatant to be tested were measured to obtain the BCA detection data, and the content of effective functional groups on the surface of the magnetic beads was calculated.

[0108] Table 2 Content of effective functional groups on the surface of magnetic beads

[0109] Example 3: The multilayer sandwich magnetic microspheres of the present invention are used for cell activation and preparation of CAR-T cells. Based on the magnetic bead concentration, take 4.0^10. 8 The corresponding volume of magnetic bead dispersion is placed in a 1.5 or 2 mL centrifuge tube. Magnetic adsorption is performed for the required time until the supernatant is clear and colorless. The supernatant is then removed. Add 1 mL PBS Buffer, vortex to mix, and centrifuge briefly to ensure no magnetic beads remain on the tube cap. Magnetically attract the tube for the required time until the supernatant is clear and colorless. Remove the supernatant and repeat this step 3 times. Take out the prepared 2 mg / mL CD3 / CD28 antibody solution, let it stand at room temperature for 15 min to restore its working temperature, add 1 mL of antibody solution to the washed magnetic beads without supernatant, vortex to mix, and centrifuge briefly to ensure that there are no magnetic beads left on the tube cap. Use a rotary mixer to seal the centrifuge tube cap at room temperature and incubate for 24 h. Remove the magnetic beads from the metal bath constant temperature shaking mixer, centrifuge briefly to ensure no magnetic beads remain on the tube cap, and magnetically attract them for the required time until the supernatant is clear and colorless, then remove the supernatant. Add 1 mL of PBS Buffer, vortex to mix, and centrifuge briefly to ensure no magnetic beads remain on the cap. Magnetically attract the tube for the required time until the supernatant is clear and colorless. Remove the supernatant and repeat this step 3 times.

[0110] Take a certain amount of the antibody-labeled magnetic beads and vortex them in a 1.5 or 2 mL centrifuge tube. Add 1 mL of PBS buffer containing 1% HSA (human serum albumin) to the centrifuge tube to rinse the magnetic beads. Place the centrifuge tube containing the resuspended magnetic beads on a magnetic rack and magnetically remove the supernatant.

[0111] Add PBMCs (peripheral blood mononuclear cells) with adjusted CD3+ T cell density to the washed magnetic beads in the specified proportion, mix well, and incubate at room temperature for 30 min. After incubation with the magnetic beads, aspirate the supernatant from the sample on a magnetic rack and wash once with 1 ml of DPBS. Aspirate the supernatant into the same tube, detect the CD3+ T cell proportion by flow cytometry, and calculate the sorting efficiency.

[0112] Resuspend the washed magnetic beads in 1 ml of complete culture medium and count them. Adjust the selected cell density to 1 × 10⁻⁶ cells using complete culture medium. 6 Count / mL, 200μL per well to be spread into a 48-well plate, minimum 4 wells.

[0113] Cells were cultured for 2 days to assess cell activation efficiency, and transfection rate was measured 4 days later. Figure 3-4 As shown, the magnetic beads labeled with CD3 / CD28 antibody exhibited good cell activation efficiency (84.7%) and transfection efficiency (54.8%).

[0114] Example 4: The multilayer sandwich epoxy magnetic microspheres of the present invention are used for cell sorting, activation, and amplification testing. Simultaneously, a certain amount of the multilayer sandwich epoxy magnetic microspheres prepared in Example 1, as well as reference standards 1, 2, and 3, were prepared for use. Reference standards 1-3 are Thermo Fisher Dynabeads™ M-450 Epoxy, epoxy magnetic beads prepared according to the scheme of Example 1 in the specification of patent CN117024788A, and epoxy magnetic beads prepared according to the scheme of Example 3 in the specification of CN114736402A, respectively.

[0115] Following the procedure in Example 3, four types of magnetic beads were labeled with CD3 / CD28 antibodies, and then the sorting efficiency, activation results, and amplification fold of the magnetic beads were compared and detected.

[0116] The sorting efficiency, activation results, and D12 amplification fold of each magnetic bead are shown in Table 3. The results show that the multilayer sandwich epoxy magnetic microspheres prepared in this application have good sorting efficiency, activation ratio, and excellent amplification fold.

[0117] Table 3. Sorting efficiency, activation results, and amplification factor results for each magnetic bead.

[0118] Magnetic bead activity detection method: 1. PBMC cell treatment: 1.1 Human PBMC cells were resuspended in PBS buffer containing 1% HSA, and samples were taken, counted, and labeled with CD3 for flow cytometry detection.

[0119] 1.2 Based on the CD3 positivity rate of PBMC cells, the CD3+ T cell density was adjusted to 1×10⁻⁶ cells using PBS buffer containing 1% HSA. 7 cells / mL.

[0120] 2. Magnetic bead cleaning: 2.1 Resuspend the magnetic beads (vortex for at least 30 seconds, or invert and mix for 5 minutes).

[0121] 2.2 According to the number of magnetic beads and the 1:1 incubation ratio of CD3+ T cells, take out the required amount of magnetic beads and transfer them to centrifuge tubes.

[0122] 2.3 Add 1 mL of PBS buffer containing 1% HSA to the centrifuge tubes, resuspend the magnetic beads, and rinse by pipetting or vortexing.

[0123] 2.4 Place the centrifuge tubes containing the resuspended magnetic beads on a magnetic rack (for magnetic beads only), let stand for 1 minute, discard the supernatant, and be careful not to pick up the magnetic beads.

[0124] 3. Magnetic bead sorting efficiency and activation efficiency testing: 3.1 Add PBMCs with adjusted CD3+ T cell density to the washed magnetic beads in proportion and mix well.

[0125] 3.2 Place the centrifuge tubes on a mixer with a rotation speed of 15-30 rpm and incubate at room temperature for 30 min.

[0126] 3.3 Place the centrifuge tubes on a magnetic rack, let them stand for 1 minute, collect the supernatant and perform counting and flow cytometry analysis, label them with CD3 to evaluate the sorting efficiency.

[0127] 3.4 Sorting efficiency formula: .

[0128] 3.5 Resuspend the mixture of magnetic beads and cells in complete culture medium containing 200 IU / mL rhIL-2, and adjust the cell density to 1×10⁻⁶. 6 cells / mL.

[0129] 3.6 After 48 hours of culture, remove the magnetic beads, transfer the cells to centrifuge tubes, place the centrifuge tubes on a magnetic rack, let them stand for 1 minute, collect the supernatant, take a small number of cells for counting, and test the expression changes of CD69+ and CD25+ on the T cell membrane to assess activation efficiency. The collected supernatant is then used for further culture.

[0130] 4. Cell D12 fold expansion detection: 4.1 Observe cell status and replenish fluids regularly. When cell density exceeds 2.5 × 10⁻⁶, [further action is needed]. 6 When the cell density reaches 0.5~1×10⁶ cells / mL or the culture medium turns yellow, adjust the cell density to 0.5~1×10⁶. 6 Cells / mL. Culture the cells for 12 days and then count the cells.

[0131] 4.2 D12 Amplification Fold Formula: .

[0132] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing multilayer sandwich epoxy magnetic microspheres, the method comprising the following steps: (1) Preparation of polyglycidyl methacrylate seed microspheres: polyglycidyl methacrylate seed microspheres were prepared from the monomer glycidyl methacrylate; (2) Preparation of PGMA-NH2 microspheres: The polyglycidyl methacrylate seed microspheres prepared in step (1) were combined with polyamines to prepare PGMA-NH2 microspheres; (3) Preparation of magnetic PGMA-NH2 microspheres: The PGMA-NH2 microspheres prepared in step (2) are further prepared into magnetic PGMA-NH2 microspheres; (4) Preparation of magnetic alkenyl microspheres: The magnetic PGMA-NH2 microspheres prepared in step (3) are reacted with glycidyl methacrylate to obtain magnetic alkenyl microspheres; (5) Preparation of multilayer sandwich carboxyl magnetic microspheres: The magnetic alkenyl microspheres prepared in step (4) are reacted with acrylic acid to obtain the multilayer sandwich carboxyl magnetic microspheres; (6) Preparation of multilayer sandwich amino magnetic microspheres: The multilayer sandwich carboxyl magnetic microspheres prepared in step (5) are reacted with polyamines to obtain the multilayer sandwich amino magnetic microspheres; (7) Preparation of multilayer sandwich alkenyl magnetic microspheres: The multilayer sandwich amino magnetic microspheres prepared in step (6) are reacted with methacrylic anhydride to obtain multilayer sandwich alkenyl magnetic microspheres; (8) Preparation of multilayer sandwich epoxy magnetic microspheres: The multilayer sandwich alkenyl magnetic microspheres prepared in step (7) are reacted with glycidyl methacrylate to obtain the multilayer sandwich epoxy magnetic microspheres.

2. The preparation method according to claim 1, characterized in that, In step (1), the polyglycidyl methacrylate seed microspheres are a polyglycidyl methacrylate seed microsphere suspension; Preferably, step (1) specifically involves: preparing PGMA seed microspheres by free radical polymerization of the monomer glycidyl methacrylate in the presence of an initiator and a dispersant; Preferably, in step (1), the initiator is selected from at least one of inorganic peroxide initiators and organic azo initiators; preferably, the inorganic peroxide initiator is selected from at least one of potassium persulfate, ammonium persulfate, sodium persulfate, etc.; preferably, the organic azo initiator is selected from at least one of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, etc. Preferably, in step (1), the dispersant is selected from one, two or more of the following: sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, polyvinylpyrrolidone or polyvinyl alcohol, etc.; Preferably, in step (1), the volume-to-mass ratio of glycidyl methacrylate to dispersant is 10-40 mL: 5-20 g; Preferably, in step (1), the volume-to-mass ratio of glycidyl methacrylate to initiator is 10-40 mL: 0.1-1 g; Preferably, step (1) is carried out in a solvent; preferably, the solvent is selected from at least one of water and ethanol; Preferably, step (1) is carried out under an inert atmosphere; Preferably, in step (1), the reaction temperature is 60-85℃; and / or the reaction time is 4-48h.

3. The preparation method according to claim 1 or 2, characterized in that, In step (2), the polyamine is selected from one, two or more of the following: ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and polyethyleneimine; Preferably, in step (2), the mass-to-volume ratio of PGMA seed microspheres to polyamine is 1 g:(10-200) mL; Preferably, step (2) specifically involves: mixing and reacting the PGMA seed microspheres prepared in step (1) with polyamine, water and surfactant to prepare PGMA-NH2 microspheres; preferably, further mixing and reacting the prepared PGMA-NH2 microspheres with polyamine and crosslinking agent to obtain the final PGMA-NH2 microspheres; Preferably, in step (2) during the preparation of PGMA-NH2 microspheres and in the final preparation of PGMA-NH2 microspheres, the polyamines are the same or different and are independently selected from one, two or more of the following: ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and polyethyleneimine; Preferably, in step (2), the crosslinking agent is selected from one, two or more of the following: epichlorohydrin, 1,4-butanediol diglycidyl ether, glycerol triglycidyl ether, and ethylene glycol diglycidyl ether; Preferably, in step (2), the surfactant is selected from one, two or more of the following: sodium 4-methyl-N-[(dodecylamino)carbonyl]benzenesulfonamide, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium dodecyl sulfonate, etc.; Preferably, in step (2) during the preparation of PGMA-NH2 microspheres, the mass-to-volume ratio of PGMA seed microspheres to polyamine is 1 g:(10-200) mL; Preferably, in step (2), the mass ratio of PGMA seed microspheres to surfactant is 1:(0.1-2); Preferably, in the final PGMA-NH2 microsphere preparation process in step (2), the mass-to-volume ratio of PGMA seed microspheres to polyamine is 1 g:(1-20) mL; Preferably, in the final PGMA-NH2 microsphere preparation process in step (2), the mass-to-volume ratio of PGMA seed microspheres to crosslinking agent is 1 g: (0.1-10) mL; Preferably, in step (2) during the preparation of PGMA-NH2 microspheres, the reaction temperature is 75-85℃; and / or the reaction time is 10-24h; Preferably, in step (2), during the final preparation of PGMA-NH2 microspheres, the reaction temperature is 15-60℃; and / or the reaction time is 2-24h.

4. The preparation method according to any one of claims 1-3, characterized in that, Step (3) specifically involves adding the PGMA-NH2 microspheres prepared in step (2) to a Fe-containing... 2+ Compounds and Fe 3+ Ammonia was added to an aqueous solution of the compound to react and obtain magnetic PGMA-NH2 microspheres. Preferably, in step (3), the reaction temperature is 70-85℃; and / or the reaction time is 0.5-2h; Preferably, in step (3), the Fe-containing 2+ The compound is selected from ferrous salts; Preferably, in step (3), the Fe-containing 3+ The compound is selected from iron salts; Preferably, in step (3), the Fe-containing 2+ Compounds and Fe-containing compounds 3+ The molar ratio of the compounds is 1:1 to 2; Preferably, in step (3), PGMA-NH2 microspheres and Fe-containing... 3+ The mass ratio of the compounds is 1:0.1-0.5; Preferably, in step (3), after adding ammonia, the pH of the reaction system is 10-11; Preferably, step (4) specifically involves: mixing and reacting the magnetic PGMA-NH2 microspheres prepared in step (3) with an organic solvent and glycidyl methacrylate to obtain magnetic alkenyl microspheres; Preferably, in step (4), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran and ethylene glycol, diethylene glycol, diethylene glycol dimethyl ether; Preferably, in step (4), the mass-to-volume ratio of magnetic PGMA-NH2 microspheres to GMA is 0.01-0.5 g: 1 mL; Preferably, in step (4), the reaction temperature is 70-85℃; and / or the reaction time is 10-24 h.

5. The preparation method according to any one of claims 1-4, characterized in that, Step (5) specifically involves: mixing and reacting the magnetic alkenyl microspheres prepared in step (4) with acrylic acid, organic solvent and initiator to obtain the multilayer sandwich-type carboxyl magnetic microspheres; Preferably, in step (5), the mass-to-volume ratio of magnetic alkenyl microspheres to acrylic acid is 1 g: 1-20 mL; Preferably, in step (5), the volume-to-mass ratio of the acrylic acid to the initiator is 1-50 mL: 1 g; Preferably, in step (5), the initiator is selected from at least one of inorganic peroxide initiators and organic azo initiators; preferably, the inorganic peroxide initiator is selected from at least one of potassium persulfate, ammonium persulfate, sodium persulfate, etc.; preferably, the organic azo initiator is selected from at least one of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, etc. Preferably, in step (5), the reaction temperature is 70-85℃; and / or the reaction time is 10-24 h; Preferably, in step (5), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran, ethylene glycol, diethylene glycol, and diethylene glycol dimethyl ether. Preferably, step (6) specifically involves: the magnetic carboxyl microspheres prepared in step (5) are mixed and reacted with polyamine, crosslinking agent, and amino protective agent in a buffer solution to obtain the multilayer sandwich-type amino magnetic microspheres; preferably, step (6) specifically involves: the magnetic carboxyl microspheres, crosslinking agent, and amino protective agent prepared in step (5) are dispersed in a buffer solution, stirred, and separated to obtain magnetic beads; then the magnetic beads are dispersed in a buffer solution, and polyamine is added to react to obtain the multilayer sandwich-type amino magnetic microspheres; Preferably, in step (6), the buffer solution is a MES buffer solution; preferably, the pH is 4.0-7.0; preferably, the concentration of the MES buffer solution is 0.1M-0.5M. Preferably, in step (6), the mass-to-volume ratio of the multilayer sandwich-type carboxylated magnetic microspheres to the polyamine is 1 g: 1-100 mL; Preferably, in step (6), the polyamine is one or more of ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polyethyleneimine, etc. Preferably, in step (6), the reaction temperature is room temperature; and / or the reaction time is 10-24 h.

6. The preparation method according to any one of claims 1-5, characterized in that, Step (7) specifically involves: mixing and reacting the multilayer sandwich-type amino magnetic microspheres prepared in step (6) with an organic solvent and acrylic anhydride to obtain multilayer sandwich-type alkenyl magnetic microspheres; Preferably, the acrylic anhydride is methacrylic anhydride; Preferably, in step (7), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran, ethylene glycol, diethylene glycol, and diethylene glycol dimethyl ether. Preferably, in step (7), the mass-to-volume ratio of the multilayer sandwich-type amino magnetic microspheres to acrylic anhydride is 1g:1-50mL; Preferably, in step (7), the reaction temperature is 70-85℃; and / or the reaction time is 10-24 h; Preferably, step (8) specifically involves: mixing and reacting the multilayer sandwich-type alkenyl magnetic microspheres prepared in step (7) with glycidyl methacrylate, an organic solvent, and an initiator to obtain the multilayer sandwich-type epoxy magnetic microspheres; Preferably, in step (8), the organic solvent is selected from any one, two or more of the following: ethanol, DMF, acetonitrile, diethylene glycol dimethyl ether, tetrahydrofuran, ethylene glycol, diethylene glycol, and diethylene glycol dimethyl ether. Preferably, in step (8), the initiator is selected from at least one of inorganic peroxide initiators and organic azo initiators; preferably, the inorganic peroxide initiator is selected from at least one of potassium persulfate, ammonium persulfate, sodium persulfate, etc.; preferably, the organic azo initiator is selected from at least one of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, etc. Preferably, in step (8), the mass-to-volume ratio of the multilayer sandwich alkenyl magnetic microspheres to glycidyl methacrylate (GMA) is 1 g: 1-50 mL; Preferably, in step (8), the mass ratio of the multilayer sandwich-type alkenyl magnetic microspheres to the initiator is 1:0.01-1; Preferably, in step (8), the reaction temperature is 70-85℃; and / or the reaction time is 10-24 h.

7. The preparation method according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Preparation of polyglycidyl methacrylate (PGMA) seed microsphere suspension: PGMA seed microsphere suspension was prepared by free radical polymerization of the monomer glycidyl methacrylate in the presence of an initiator and a crosslinking agent; (2) Preparation of PGMA-NH2 microspheres: The PGMA seed microsphere suspension prepared in step (1) is mixed with polyamine, water and surfactant and reacted to swell and amination in one step to prepare amino microspheres; then the obtained amino microspheres are mixed with polyamine, water and crosslinking agent to obtain PGMA-NH2; (3) Preparation of magnetic PGMA-NH2 microspheres: The PGMA-NH2 microspheres prepared in step (2) are added to Fe-containing... 2+ Compounds and Fe 3+ Ammonia was added to an aqueous solution of the compound to react and obtain magnetic PGMA-NH2 microspheres. (4) Preparation of magnetic alkenyl microspheres: The magnetic PGMA-NH2 microspheres prepared in step (3) are mixed with organic solvent and glycidyl methacrylate to obtain magnetic alkenyl microspheres; (5) Preparation of multilayer sandwich carboxyl magnetic microspheres: The magnetic alkenyl microspheres prepared in step (4) are mixed and reacted with acrylic acid, organic solvent and initiator to obtain the multilayer sandwich carboxyl magnetic microspheres; (6) Preparation of multilayer sandwich amino magnetic microspheres: The magnetic carboxyl microspheres prepared in step (5) are mixed and reacted with polyamine, EDC and NHS in a buffer solution to obtain the multilayer sandwich amino magnetic microspheres; (7) Preparation of multilayer sandwich alkenyl magnetic microspheres: The multilayer sandwich amino magnetic microspheres prepared in step (6) are mixed with organic solvent and acrylic anhydride to react and obtain multilayer sandwich alkenyl magnetic microspheres; (8) Preparation of multilayer sandwich epoxy magnetic microspheres: The multilayer sandwich alkenyl magnetic microspheres prepared in step (7) are mixed and reacted with glycidyl methacrylate, organic solvent and initiator to obtain the multilayer sandwich epoxy magnetic microspheres.

8. The multilayer sandwich epoxy magnetic microspheres prepared by the preparation method according to any one of claims 1-7; Preferably, the Z-average particle size of the multilayer sandwich epoxy magnetic microspheres is 1000nm-8000nm; Preferably, the saturation magnetic strength (VSM) of the multilayer sandwich epoxy magnetic microspheres is 30 emu / g-50 emu / g; Preferably, the magnetic attraction time of the multilayer sandwich epoxy magnetic microspheres is 1s-5s; Preferably, the effective functional group content of the surface of the multilayer sandwich epoxy magnetic microspheres is 1.0^10. -11 μmol / beads - 6.0^10 -11 μmol / beads.

9. The antibody-labeled multilayer sandwich epoxy magnetic microspheres of claim 8; Preferably, the antibody is a CD3 / CD28 antibody.

10. Any of the following applications of the multilayer sandwich epoxy magnetic microspheres prepared by the preparation method according to any one of claims 1-7, the multilayer sandwich epoxy magnetic microspheres according to claim 8, and the antibody-labeled multilayer sandwich epoxy magnetic microspheres according to claim 9: (i) Applications in cell sorting, protein / antibody separation and purification, nucleic acid separation and purification, and immunoassay / detection; (ii) Application in the preparation of reagents for cell sorting, protein / antibody separation and purification, nucleic acid separation and purification, and immunoassay / detection; Preferably, the applications of cell sorting include stem cell isolation, tumor cell detection, immune cell therapy, and pathogen enrichment.

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